Metody badania RNA w neuronach

(1)

Magdalena Dziembowska

Metody badania RNA w neuronach

(2)

A Golgi-stained pyramidal cell in the parietal cortex of a rat. The high power images at the right show dendritic spines on apical and basilar dendritic branches. Photo by Grazyna Gorny

T cell

fibroblast

(3)

Polyribosomes localized in dendritic spines

Steward and Levy w 1982

(4)

Fig. 1: A segment of pyramidal cell dendrite from stratum radiatum (CA1) with thin, stubby, and mushroom-shaped spines. Spine synapses colored in red, stem (or shaft) synapses colored in blue. The dendrite was made transparent in the lower image to enable visualization of all synapses. Photo by Josef Spacek.

Synaptic spines are „structural units” that undergo plastic changes in

response to synaptic stimulation

(5)

Local mRNA translation in dendritic spines

(6)

A model of Arc-dependent LTP consolidation in the dentate gyrus

(7)

MAP2 GFAP

Metody wizualizacji mRNA w neuronach

(8)

In situ hybrydyzacja z sonda RNA wyznakowana digoksygeniną

(9)

(10)

High-resolution fluorescent in situ hybridization procedure to comprehensively evaluate mRNA localization dynamics during early Drosophila embryogenesis.

Lecuyer et al. 2007

(11)

„Single molecule FISH” to metoda in situ hybrydyzacji pozwalająca na obrazowanie pojedynczej cząsteczki mRNA w komórce dzięki wykorzystaniu wielu fluorescencyjnie wyznakowanych sond zaprojektowanych do rozpoznawania sekwencji

w obrębie tej samej cząsteczki mRNA

Przy użyciu tej metody można np. jednocześnie wykrywać dwa różne mRNA w komórce lub mRNA postałe w wyniku fuzji 2 transkryptów (translokacji genomowych)

jak np. BCR-ABL

(12)

Obrazowanie mRNA w dendrytach komórek nerwowych z wykorzystaniem smFISH

(13)

Activity-dependent local translation of MMP-9

Dziembowska and Wlodarczyk, Int J Biochem Cell Biol , 2012

Dziembowska et al., J Neuroscience 2012

(14)

Medial perforant path LTP - a well established model of synaptic plasticity

(15)

In situ hybridization shows increse in MMP-9 expression in granular layer and

molecular layer of dentate gyrus 2h after medial perforant path LTP

(16)

MMP-9 in situ hybridization Arc in situ hybridization

(17)

Figure 2 from Michael Doyle and Michael A Kiebler The EMBO Journal online publication

doi:10.1038/emboj.2011.278

Sushi belt model

(18)

MS2 system to stain targeted mRNA in the living cell

Martha S. Rook, Mei Lu, and Kenneth S. Kosik

(19)

MS2 system to stain targeted mRNA in the living cell

GFP GFP GFP GFP GFP

Dziembowska et al., J Neurosci. 2012

(20)

MMP-9 protein is enriched in the synaptoneurosomal fraction

Homogenate (H)

Hippocampus or cortex

Filtrate (F)

Cytosol (C) HOMOGENIZATION

FILTRATION

(PVDF 100, 60, 30, 10 μm)

CENTRIFUGATION (1000 rpm, 30 min)

Synaptoneurosomes (SN)

Washing and centrifugation

PSD-95

GFAP c-Jun

GAPDH MMP-9

H F SN C

1. Polyadenylation of MMP-9 in response to synaptic stimulation 2. Isolation of polyribosomes

3. Direct study of MMP-9 protein synthesis by Click-iT chemistry

(21)

Synaptoneurosomy w mikroskopie elektronowym

(22)

PAT assay

(23)

MMP-9 polyadenylation measured by PAT ssay in synaptoneurosomes after glutamate stimulation

MMP-9 CDS 3’ UTR

STOP PolyA I CPE I CPE II PolyA II PolyA III

2500 CCCUUUUAUUUAUUAUGU 2517 2592 CCCUUUUAUUUAUUAUGU 2609 2416 ACCUUUUGUUUUUAUGGG 2433 2502 ACCUUUUAUUUUUGUGUG 2519 R. norvegicus

M. musculus R. norvegicus M. musculus CPE I

CPE II

A

B

(24)

Polysomal fractionation

(25)

J. Miłek

Polyribosome association of MMP-9 mRNA in synaptoneurosomes

(26)

Local synthesis of MMP-9 protein detected with click chemistry

Synaptoneurosomes

Incubation with glycine analogue (HPG) and stimulation

Protein isolation (lysis in 1% SDS)

And Click-iT reaction with AHA tagged with biotin Precipitation on streptavidin dynabeads

Western blot with anti-MMP-9 antibody

(27)

Fragile X syndrome

is the most common form of inherited mental retardation in males and is also a significant cause of mental retardation in females. It affects about 1 in 4,000 males and 1 in 8,000 females. Nearly all cases of fragile X syndrome are caused by an alteration (mutation) in the FMR1 gene where a DNA segment, known as the CGG triplet repeat, is expanded.

Badanie oddziaływania białko-RNA

(28)

One of the characteristic features of FXS neurons is the abnormal dendritic spine morphology (Rudelli 1985).

Immature, long and thin spines are also observed in FMR1 KO mice, a model of FXS.

Malformations of the dendritic spines may lead to cognitive disorders

Fmr1 KO WT

(29)

anti-FMRP antibody or normal IgG

Dynabeads A Synaptoneurosome

extract

Precleared

extract Antibody-bound

beads

2. RNA isolation and RT-PCR 1. Western blot

Coimmunoprecipitation of FMRP protein with MMP-9 mRNA

Immunoprecypitated complexes

(30)

MMP-9 mRNA is precipitated with anti-FMRP antibodies

Janusz et al., J Neuroscience 2013

(31)

Janusz et al., J Neuroscience 2013

Polysomal fraction isolated from WT and Fmr1KO mice

before and after stimulation with DHPG

(32)

Nature Medicine Volume:17,Pages:1353–1355 (2011)

MMP-9

Local translation of MMP-9 is regulated by FMRP

minocycline

(33)

Dziembowska i wsp., Am J Med Genet A. 2013